Clostridium difficile is a leading cause of hospital-acquired illness in developed countries. Treating C. difficile infection (CDI) is complicated by antibiotic resistance, recurrence and hypervirulence of emerging strains that produce a highly toxic variant of TcdB. TcdB is a major C. difficile virulence factor that contributes to gastrointestinal damage. Our work has focused on characterizing the differences between the TcdB1 (low toxicity) and TcdB2 (high toxicity) forms of TcdB. TcdB2 enters cells more rapidly and with a higher efficiency than TcdB1 and this correlates with differences in cytotoxicity. We recently found that the 1769-1787 region of TcdB2 differs from TcdB1 at 6 residues and a peptide from this region can facilitate uptake of TcdB and other proteins. TcdB2 exhibits many of the characteristics of cell penetrating peptides (CPP) and this activity is likely to account fo the toxin's more rapid cell entry compared to TcdB1. This is the first example of a CPP derived from an intracellular bacterial toxin and may represent an entirely new area of study. As such these studies will address two specific aims. SA1: Determine the Basic Characteristics of TcdB2's Mechanism of Action and SA2: Characterize the Role of TcdB in TcdB Variability. In the first specific aim, we will assess the basic characteristic of TcdB2 and determine which residues are critical to the CPP activity of this sequence. In parallel experiments we will determine how TcdB promotes cell entry of a variety of proteins. Experiments in aim 2 will determine how variation in the 1769-1787 sequence contributes to the overall differences in toxicity between TcdB1 and TcdB2.